The objective of the proposed research is to contribute to the understanding of the molecular mechanisms of prion replication. Investigation of prion pathogenesis advances knowledge that could benefit sufferers of Creutzfeldt-Jakob and other prion diseases. Such work also enhances our insight into mechanisms of protein aggregation, which occurs in Alzheimer's, Parkinson's, and other neurodegenerative conditions. The infectious prion is largely or entirely composed of PrPSc, the misfolded isoform of the normal cellular protein PrPC. This project seeks to examine the role of polybasic domains (PBDs) in conversion of PrPC to PrPSc, the pivotal event in prion replication. Previous studies indicate that PBDs of PrPC may serve as binding sites for the misfolded isoform PrPSc. This application proposes to determine the role of PBDs in both PrPC-PrPSc binding and in the autocatalytic conversion of PrPC by PrPSc. Specifically, the proposed study will determine if the PrPC PBDs, alone or in combination, mediate interaction with PrPSc and facilitate conversion. The methods involve co-immunoprecipitation to examine binding and in vitro protein misfolding cyclic amplification (PMCA) to examine autocatalytic conversion. The co-immunoprecipitation method will determine if antibody-bound PrPC can bind PrPSc under various conditions. The PMCA method, a sensitive and specific assay, will test the ability of various PrPC preparations to undergo conversion to protease-resistant autocatalytic PrPSc. Two techniques will be employed to assess the role of the PBDs. First, PBD-deleted PrPC, generated in cultured neuronal cells and purified, will be tested by the above methods for its ability to bind PrPSc and to be converted into nascent PrPSc. Second, PBD peptides will be examined for their ability to competitively inhibit PrPSc binding to PrPC and the subsequent conversion process. Results of these studies will indicate the role of PBDs in PrPC conversion, potentially identifying the binding site(s) on PrPC for its conversion to PrPSc. The peptide inhibition experiments will also evaluate a potential therapeutic avenue for inhibition of prion replication.